Project Summary/Abstract The accumulation of pathological tau is the main component of neurofibrillary tangles in Alzheimer?s disease and several degenerative diseases, referred to as tauopathies. We previously found that administration of an anti- tau antibody to human tau (h-tau) or expression of an anti-tau secreted single-chain variable fragment (scFv) in the central nervous system (CNS) of h-tau transgenic mice (P301S-tg) decreased but did not remove all tau- associated pathology. While these and other studies demonstrate immunotherapeutic approaches targeting tau can influence tau pathogenesis, conventional immunotherapeutic approaches present some limitations that preclude their full potential when targeting tauopathy. Including, immunotherapy is limited to targeting extracellular proteins whereas the majority of pathological tau remains in the cytosol of cells, not typically accessible to an extracellular antibody or secreted scFv. In addition, a significant limitation of passive immunization is the necessity for chronic administration of antibodies of which only a small % (0.1-0.2%) cross the blood-brain-barrier (BBB). When translated into humans, this may be limited by manufacturing. To potentially overcoming these limitations, we hypothesize the expression of anti-tau scFv in the cytosol of neurons (intrabodies) will reduce tauopathy with improved efficacy. To enhance the ingenuity of conventional intrabodies, we have engineered chimeric anti-tau intrabodies fused to ubiquitin harboring distinct mutations or containing a heat-shock motif (HSC) with the goal of shuttling intracellular tau for degradation by either the proteasome, lysosome or chaperone-mediated autophagy (CMA). In preliminary data, expressing the modified anti-tau intrabodies in primary neuronal cultures expressing h-tau reduced h-tau protein levels. Moreover, the expression of the modified anti-tau intrabodies in aged P301S-tg mice after disease onset effectively reduced tauopathy whereas; a conventional anti-tau intrabody containing no tags was ineffective in reducing tauopathy. The goals for this project are to validate the degradation mechanisms by which the anti-tau intrabodies reduce tau levels and determine the extent to which anti-tau intrabodies prevent or stop tauopathy. In addition, we aim at bypass the BBB by combining our anti-tau intrabodies with the recent advances in adeno-associated virus-mediated gene transfer that provide global-neuronal transduction in the adult mouse CNS by intravenous administration. We further propose to generate new anti-tau intrabodies that target aberrant phosphorylated tau sites, which may display an enhanced efficacy by selectively degrading pathological tau. The current proposal harnessed the strength of intrabodies, which are amendable for targeting specific domains or modifications with the cell-intrinsic mechanisms that regulate protein degradation potentially providing a new immunotherapeutic agent with improved efficacy.